Prion diseases are amyloidoses, like Type 2 diabetes and Alzheimer's disease, but with the important distinction of being infectious. In these diseases accumulation of amyloid fibers of misfolded protein is associated with tissue pathology. We use yeast as a model system to study how cellular factors influence amyloid propagation. The yeast [PSI] prion is a cytoplasmic amyloid of the translation termination factor Sup35p. The disaggregating activity of the protein chaperone Hsp104, which is thought to break prion aggregates or "seeds" into more numerous pieces, is required for yeast prion propagation. Protein disaggregation by Hsp104 requires assistance of Hsp70 and its co-chaperone Hsp40. Our previously isolated Hsp70 mutant (Ssa1-21p) impairs [PSI] propagation. Suppressor analysis of Ssa1-21p identified Hsp70 residues involved in interactions with co-chaperones. We found that a subset of co-chaperones affected [PSI] propagation by regulating Hsp70. These co-chaperones also regulate Hsp90, an essential protein chaperone that, among other activities, functions with Hsp70 in a folding pathway for many "client" proteins. We earlier showed Hsp90 does not affect [PSI], suggesting that these co-chaperones influence [PSI] only through Hsp70. Mutational analysis of Sti1p (Hop1 in humans), a major Hsp90 co-chaperone that coordinates Hsp70 and Hsp90 functions, showed that separable domains of Sti1p regulated Hsp70 and Hsp90 but intact Sti1p was necessary for client protein folding. This work was the first to demonstrate that Sti1p independently regulates Hsp70 and Hsp90 and that physical linking of the two chaperones by Sti1p was necessary for proper function of the client folding pathway. We further showed that human Hop1 was conserved in regulation of Hsp70 regarding [PSI] propagation and in ability to independently regulate Hsp70 and Hsp90. We constructed the first Sup35-GFP fusion protein that functions normally in both translation termination and prion propagation and used it to show that SSA1-21 [PSI+] cells have larger Sup35p aggregates than wild type cells, suggesting that Ssa1-21p interferes with disaggregation of prion aggregates. We also found that numbers of Sup35p aggregates in [PSI+] cells did not correlate with numbers of prion seeds determined genetically, suggesting that much aggregated Sup35p does not function as prion seed or that current methods for estimating seed numbers are unreliable. We earlier showed that millimolar guanidine in growth medium inactivates Hsp104 and thereby cures yeast of prions. After guanidine-inactivation of Hsp104 arrests prion seed replication, curing is believed to require cell division to dilute the seeds among progeny until there are too few to distribute to daughter cells. The number of prion seeds in yeast cells has been estimated by counting the number of cell divisions required to produce cells lacking prions after guanidine addition. Our monitoring of Sup35-GFP fluorescence after guanidine addition revealed a biphasic response of first increased Sup35 aggregation and then dissolution of aggregates. After arresting cell division, we found that guanidine still caused complete dissolution of Sup35 aggregates and appearance of prion-free cells, meaning that prion seed numbers cannot be determined as just described, and that an as yet unknown process eliminates the prion aggregates. Current work includes biochemical analysis to determine how chaperone mutations affect enzymatic activities as well as physical and functional interactions of chaperones with each other and with amyloid, and genetic analysis to identify processes that cause prion elimination after guanidine treatment.